Sex-specific differences in systemic immune responses in MIS-C children

Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare manifestation of Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) infection that can result in increased morbidity and mortality. Mounting evidence describes sex disparities in the clinical outcomes of coronavirus disease 2019 (COVID-19). However, there is a lack of information on sex-specific differences in immune responses in MIS-C. This study is an observational and cross-sectional study and we wanted to examine immune parameters such as cytokines, chemokines, acute phase proteins (APPs), growth factors, microbial translocation markers (MTMs), complement components and matrix metalloproteinases (MMPs) in MIS-C children, based on sex. Male children were associated with heightened levels of pro-inflammatory cytokines—IFNγ, IL-2, TNFα, IL-1α, IL-1β, IL-6, IL-12, G-CSF and GM-CSF, chemokines-CCL2, CCL11, CXCL1, CXCL8 and CXCL10, acute phase proteins-α-2M, CRP, growth factors VEGF and TGFα, microbial translocation markers- iFABP, LBP, EndoCAb, complement components—C1q, MBL and C3 and matrix metalloproteinases MMP-8 and MMP-9 compared to female children with MIS-C. These results indicate that the heightened immune response in males is a characteristic feature of MIS-C. These findings might explain the differential disease pathogenesis in males compared to females with MIS-C and facilitate a deeper understanding of this disease.

To delineate the immune responses in males and females with MIS-C, we performed a detailed analysis of the sex differences in immune responses by assessing the levels of plasma cytokines, chemokines, acute phase proteins, growth factors, microbial translocation markers, complement proteins and matrix metalloproteinases.Our results indicate that the heightened immune responses in males lead to an exaggerated inflammatory response which could potentially predispose male children to hyperinflammation and poor innate and adaptive immune responses.

Basic characteristics
We measured an array of immunological parameters on stored plasma samples of 65 MIS-C children hospitalized from June to September 2020.As shown in Tables 1 and 2, the median age was 5.8 years (range 1 month to 14 years), 54% (35/65) were males and 46% were females.All MIS-C children were seropositive (IgG) among whom 29% of males and 23% of females had a severe disease that required PICU care.Among the laboratory parameters, WBC, and lymphocyte levels were significantly increased in males compared with females in MIS-C children.Among the biochemical parameters analysed, the levels of CRP, ferritin and Troponin-I levels were significantly higher in males when compared with female MIS-C children.

Heightened plasma levels of cytokines are associated with male MIS-C children
We wanted to examine the influence of sex on the levels of cytokine responses in MIS-C children.Therefore, we measured the levels of cytokines (IFNγ, IL-2, TNFα, IL-1α, IL-1β, IFNα, IFNβ, IL-6, IL-12, G-CSF, GM-CSF and IL-17A) in male and female MIS-C children.As shown in Fig. 1, we observed that male MIS-C children exhibited significantly increased levels of IFNγ (GM of 47.24 pg/ml in males compared to 29.44 pg/ml in females; p = 0.0043), IL-2 (GM of 54.92 pg/ml in males compared to 38.19 pg/ml in females; p = 0.0193), TNFα (GM of 80.25 pg/ml in males compared to 56.93 pg/ml in females; p = 0.0200), IL-1α (GM of 93.67 pg/ml in males compared to 52.45 pg/ml in females; p < 0.0001), IL-1β (GM of 12.56 pg/ml in males compared to 10.19 pg/ml in females; p = 0.0068), IL-6 (GM of 251.8 pg/ml in males compared to 138 pg/ml in females; p = 0.0053), IL-12 (GM of 60.09 pg/ml in males compared to 35.72 pg/ml in females; p = 0.0008), G-CSF (GM of 61.35 pg/ml in males compared to 32.77 pg/ml in females; p < 0.0001) and GM-CSF (GM of 49.53 pg/ml in males compared to 33.56 pg/ml in females; p = 0.0006) compared to female MIS-C children.Thus, male MIS-C children have exacerbated pro-inflammatory cytokine responses compared to female MIS-C children.

Heightened plasma levels of CC and CXC chemokines are associated with male MIS-C children
Next, we wanted to examine the influence of sex on the levels of chemokines in MIS-C children.Therefore, we measured the levels of chemokines (CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL20, CXCL1, CXCL8, CXCL10 and CX3CL1) in male and female MIS-C children.As shown in Fig. 2, we observed that male MIS-C children exhibited significantly increased levels of CCL2 (GM of 2952 pg/ml in males compared to 792.3 pg/ml in females; p = 0.0001), CCL11 (GM of 453.9 pg/ml in males compared to 274.5 pg/ml in females; p = 0.0038),

Heightened plasma levels of acute phase proteins, growth factors and microbial translocation markers are associated with male MIS-C children
Further, we wanted to examine the influence of sex on the levels of acute phase proteins and microbial translocation markers in MIS-C children.Therefore, we measured the levels of acute phase proteins α-2-M, CRP, SAP, and Hp, growth factors (VEGF and TGFα) and microbial translocation markers (LPS, sCD14, iFABP, LBP and EndoCAb) in male and female MIS-C children.As shown in Fig. 3, we observed that male MIS-C children  exhibited significantly elevated levels of α-2-M (GM of 716.8 pg/ml in males compared to 254.1 pg/ml in females; p = 0.0008) and CRP (GM of 174.6 pg/ml in males compared to 52.43 pg/ml in females; p = 0.0005), growth factors VEGF (GM of 417.4 pg/ml in males compared to 248.3 pg/ml in females; p = 0.0127), TGFα (GM of 149.6 pg/ ml in males compared to 92.47 pg/ml in females; p = 0.0282) and microbial translocation markers, LPS (GM of 0.09817 pg/ml in males compared to 0.06585 pg/ml in females; p = 0.0120), iFABP (GM of 2259 pg/ml in males compared to 1574 pg/ml in females; p = 0.0138), LBP (GM of 105.2 pg/ml in males compared to 81.04 pg/ml in females; p = 0.0097), and EndoCAb (GM of 2.694 pg/ml in males compared to 1.232 pg/ml in females; p = 0.0127) compared to female MIS-C children.Thus, male MIS-C children have exacerbated acute phase protein, growth factor and microbial translocation marker responses compared to female MIS-C children.
Heightened plasma levels of complement components and complement regulators are associated with male MIS-C children Next, we wanted to examine the influence of sex on the levels of complement components and complement regulators in MIS-C children.Therefore, we measured the levels of complement proteins like C1q, C2, C3, C3b/ iC3b, C4, C4b, C5, C5a, MBL and complement regulatory proteins like Factor B, Factor D, Factor H and Factor I in male and female MIS-C children.As shown in Fig. 4, we observed that male MIS-C children exhibited significantly elevated levels of C1q (GM of 31.89pg/ml in males compared to 29.33 pg/ml in females; p = 0.0080), MBL (GM of 71.93 pg/ml in males compared to 49.24 pg/ml in females; p = 0.0004), and C3 (GM of 145 pg/ ml in males compared to 118.9 pg/ml in females; p = 0.0086) compared to female MIS-C children.Thus, male MIS-C children have exacerbated levels of complement components and complement regulators compared to female MIS-C children.

Heightened plasma levels of matrix metalloproteinases are associated with male MIS-C children
Next, we wanted to examine the influence of sex on the levels of matrix metalloproteinases in MIS-C children.Therefore, we measured the levels of matrix metalloproteinases like MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP13 in male and female MIS-C children.As shown in Fig. 5, we observed that male MIS-C children exhibited significantly increased levels of MMP8 (GM of 32,472 pg/ml in males compared to 24,870 pg/ml in females; p = 0.0004) and MMP9 (GM of 35,921 pg/ml in males compared to 26,836 pg/ml in females; p = 0.0011) compared to female MIS-C children.Thus, male MIS-C children have exacerbated levels of matrix metalloproteinases compared to female MIS-C children.

Immune parameters can strongly discriminate sex-based immune responses in MIS-C children
Using immunological parameters like cytokines, chemokines, acute-phase proteins, growth factors, microbial translocation markers, complement components, and MMPs, we performed PCA (principal component analysis) plot computing normalised immune markers after excluding those factors with commonalities as low as 0.5 (Fig. 6A) in order to determine the discriminatory ability of immune parameters between male and female of MIS-C children.Additionally, we used data sets from two groups of kids to do hierarchical clustering analysis   www.nature.com/scientificreports/utilising cytokines, chemokines, acute-phase proteins, growth factors, microbial translocation indicators, complement components, and MMPs.For cellular subgroups, a heatmap and dendrogram are shown (Fig. 6B).Ward's supervised clustering algorithm and index were used to create the dendrogram, which is demonstrated to be capable of discrimination.

Discussion
In this study, we aimed to determine the influence of sex on immune responses in MIS-C children.We observed that male children with MIS-C exhibited significantly elevated levels of pro-inflammatory cytokines, chemokines, growth factors, microbial translocation markers (MTMs) and Matrix Metalloproteinases (MMPs) when compared with female MIS-C children.Increasing evidence that the male sex is a risk factor for COVID-19 mortality and more severe illness.Our data indicate significant disparities in the immune responses in male and female children with MIS-C and suggest a possible immunological underpinning of the different means of disease progression among sexes.These data also offer a possible basis for taking sex-dependent methods for prognosis, prevention, care, and therapy for the patient with MIS-C.It has been established that the immune system differs across the sexes and is varied in terms of both innate and adaptive immunity 19 .Biological sex has an impact on innate and adaptive immune responses to self and foreign antigens, resulting in sex disparities in autoimmunity, and responses to infections and vaccines 20 .The activity of X-linked genes can be used to explain the discrepancy in the impact of sex-specific diseases.There are at least two known variables that affect sexual dimorphism's impact on immunity: hormonal changes and heredity, namely the X chromosome.Some of the immune response-related proteins encoded by genes on the X chromosome include toll-like receptors and interleukins 19 .Additionally, the sex-specific expressions of TMPRSS2 may help to partially explain the COVID-19 pandemic's male preponderance 21 .Some variations can also be attributed to variations in the activity and expression of the human angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV-2 9 .
Past studies have shown that cytokines and chemokines were significantly elevated and have a role in the pathogenesis of the MIS-C 3,22,23 .In an adult SARS-CoV-2 study, adult females exhibited higher levels of interferon-α (IFNα) than adult males 24 .On the contrary, a study revealed that male patients exhibited elevated plasma levels of innate immune cytokines such as IL-8, IL-18, and the chemokine CCL5, along with a pronounced induction of monocytes.In contrast, female patients demonstrated a more robust activation of T cells.The study further observed that a deficient T-cell response was negatively correlated with age and led to a more adverse disease outcome in male patients, with no corresponding impact on female patients 25 .Similarly, we observed that male MIS-C children had significantly elevated plasma levels of cytokines and chemokines compared with females.Sex differences in cytokines and chemokines responses to MIS-C in children warrant further investigation in larger cohorts of children.
In MIS-C and COVID-19, CRP levels have been used as a marker of disease pathology 17 .Previously, we have shown that CRP has a major contribution to the pathogenesis of this disease entity in MIS-C and acute COVID-19 children 26 .In this study, we observed male MIS-C children had elevated levels of CRP and a-2-M compared with female MIS-C children, which indicates that male children had higher levels of pathogenesis compared with female children.In the absence of overt bacteremia, microbial translocation is the process through which microbial products like lipopolysaccharide (LPS) and bacterial DNA move from the intestinal lumen to the systemic circulation 27 .Our earlier data determined that MIS-C children had elevated levels of LPS, LBP, and sCD14, all markers of gut damage and microbial translocation across the gastrointestinal tract 26 .Microbial translocation also plays a role in gut microbiome dysbiosis in COVID-19 28 .Recent data revealed that zonulin, a biomarker of intestinal permeability found in children with MIS-C in the GI tract, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation 29 • Myocarditis and/or pericarditis represent infrequent adverse cardiac events observed in adolescent and young adult males following SARS-CoV-2 mRNA vaccination.These events are characterized by acute systemic inflammation, elevated troponin and BNP levels, and abnormalities in cardiac imaging, as documented by Katoto et al.and Anis Barmada et al. 30,31 .Notably, a recent study by Ulucay AS reported a higher susceptibility to myocarditis (both associated with COVID-19 and mRNA vaccines) among males (0.09%) compared to females (0.04%) 32 .In our study, we observed that male children had elevated levels of LPS, iFABP, LBP and EndoCAb compared with female MIS-C children.These results imply that male children could be affected by higher levels of gut damage and microbial translocation across the gastrointestinal tract than female children.
The innate immune system's complement system is an essential component that contributes to the pathogenesis of COVID-19.In an adult healthy Caucasian population study, Alternative Pathway activity, C3, and MBL levels were found to be lower in females compared to males while factor D concentrations were higher 19 .Recently, there have been reports that the dysregulation of complement proteins are involved in the COVID-19 disease pathology in adults and children [33][34][35][36] .The immunological characteristics of MIS-C have been shown to include the activation of complement pathways and modification of innate and adaptive immune responses 37 .Previous reports indicated that MIS-C and acute COVID-19 children were associated with highly elevated levels of activation markers of the classical, alternative and terminal pathways 38,39 .Children with SARS-CoV-2 infection exhibited elevated levels of sC5b9, which correlated with disease severity 35,38,40,41 .A recent study with MIS-C children on complement observed elevated levels of activation markers of the classical, alternative and terminal pathways 38 .Our data determine that males have significantly higher complement activity and levels of complement components compared to females.Moreover, our results imply that sex should be considered in complement-associated pathology and also in complement-directed therapies.
Matrix metalloproteinases (MMPs) are important zinc-dependent endo proteases involved in both physiological and pathological ECM remodelling 42 .Increased plasma levels of MMP-9 were found in patients with severe www.nature.com/scientificreports/ARDS 43 and COVID-19 patients 44 .Another study described the immune-based signature of COVID-19 patients, relating serum MMP-9 levels with the severity and as a biomarker of COVID-19 45,46 .It has been observed that in lung tissue from COVID-19 patients, the MMP9 gene is up-regulated, and the protein contributes to cytokine recruitment 47 .We have previously observed that MMPs play a role in the pathogenesis of MIS-C and acute COVID-19 in children 48 .In the current study, we observed male MIS-C children exhibited elevated levels of MMP-8 and MMP-9 which is associated with disease severity and pathogenesis of MIS-C.These results indicate that male children may have more severity and pathology of MIS-C than female children.
Our study has certain limitations, such as the fact that we only looked at children from one location, the limited number of samples, and the lack of longitudinal follow-up data.Our work, on the other hand, breaks new ground by analysing a wide range of immunological measures in both male and female MIS-C children and by offering a more in-depth investigation of inflammatory markers.In conclusion, our work is the first to provide a thorough analysis of sex-based disparities in the immunological parameter profile of Indian children with MIS-C.The fact that sex differences are a significant contributor to individual differences makes understanding them vital.The efficacy of clinical practice, health policy, and treatments will be increased by having a better understanding of the disparities in outcomes between male and female patients.Future studies are necessary to comprehend the reasons for the sex gap, and they may also be of relevance to public health decision-makers.

Study design and participants
We prospectively enrolled children admitted to Kanchi Kamakoti CHILDS Trust Hospital (KKCTH), Chennai, India Institute of Child Health, Dr Mehta's Children Hospital, Rainbow Children's Hospital, from 1 June 2020 to 30 September 2020 with MIS-C children.The study population and the enrolment criteria have been previously described 26,48,49 .Briefly, we included children of either sex between 1 month and 14 years of age or whose parents were willing to provide informed consent/assent.Blood collection was performed prior to any immunomodulatory medication.Plasma was isolated and used for measuring multiple immune parameters.The demographic, epidemiological, medical and laboratory data have been previously reported 26,48,49 and are described in Table 1.Children with MIS-C were diagnosed according to the World Health Organisation (WHO) case definition 50 and all the enrolled MIS-C cases have no other microbial or viral inflammatory focus.Blood was collected in EDTA tubes (BD Biosciences) or heparin tubes and processed within 4 h of collection at the National Institute for Research in Tuberculosis (NIRT), Chennai.To avoid measurement bias and to increase the precision of the estimates for the accuracy of the assay, the study staff involved in immunological assays were blinded to any clinical data.

Measurement of acute phase proteins
Plasma levels of alpha-2 macroglobulin (α 2 M), C-reactive protein (CRP), haptoglobin (Hp), and serum amyloid P (SAP), using a Milliplex MAP Human CVD Panel Acute Phase magnetic bead panel 3 from Millipore, were measured using a multiplex platform according to the manufacturer's instructions.The lowest detection limits for acute phase proteins were as follows: α-2-M, 0.49 ng/mL; CRP, 0.05 ng/mL; Hp, 0.06 ng/mL; and SAP, 0.06 ng/mL.

Figure 2 .
Figure 2. Heightened plasma levels of CC and CXC chemokines are associated with male MIS-C children.The plasma levels of CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL20, CXCL1, CXCL8, CXCL10 and CX3CL1 were measured in male MIS-C (n = 35), and female MIS-C children (n = 30).The data are represented as Box and Whiskers plots.p values were calculated using the Mann-Whitney U test.

Figure 3 .
Figure 3. Heightened plasma levels of acute phase proteins, growth factors and microbial translocation markers are associated with male MIS-C children.The plasma levels of C-reactive protein (CRP), alpha-2 macroglobulin (α-2 M), haptoglobin, Serum Amyloid P (SAP), VEGF, TGFα, LPS, lipid-binding protein (LBP), endotoxin core antibodies IgG (EndoCAb), intestinal fatty acid binding protein (iFABP) and sCD14 were measured in male MIS-C (n = 35), and female MIS-C children (n = 30).The data are represented as Box and Whiskers plots.p values were calculated using the Mann-Whitney U test.

Figure 4 .
Figure 4. Heightened plasma levels of complement components and complement regulators are associated with male MIS-C children.The plasma levels of complement components (C1q, C2, C3, C4, C4b, MBL, C5), regulators (factor B, factor D, factor H and factor I) and activation products (iC3b, C5b) were measured in male MIS-C (n = 35), and female MIS-C children (n = 30).The data are represented as Box and Whiskers plots.p values were calculated using the Mann-Whitney U test.

Figure 5 .
Figure 5. Heightened plasma levels of matrix metalloproteinases are associated with male MIS-C children.The plasma levels of Matrix Metallo Proteinases (MMP) MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12 and MMP13 were measured in male MIS-C (n = 35), and female MIS-C children (n = 30).The data are represented as Box and Whiskers plots with each circle representing a single individual.p values were calculated using the Mann-Whitney U test.

Table 2 .
MIS-C children's clinical parameters of the study population.
. MIS-C children's demographic and biochemical parameters.Vol:.(1234567890)malescompared to 19,960 pg/ml in females; p = 0.0320) compared to female MIS-C children.Thus, male MIS-C children have exacerbated chemokine responses compared to female MIS-C children.